A comparative assessment of the acute inhalation toxicity of vanadium compounds.

Vanadium compounds have become important in industrial processes, resulting in workplace exposure potential and are present in ambient air as a result of fossil fuel combustion. A series of acute nose-only inhalation toxicity studies was conducted in both rats and mice in order to obtain comparative data on the acute toxicity potential of compounds used commercially. V2O3, V2O4, and V2O5, which have different oxidation states (+3, +4, +5, respectively), were delivered as micronized powders; the highly water-soluble and hygroscopic VOSO4 (+4) could not be micronized and was instead delivered as a liquid aerosol from an aqueous solution. V2O5 was the most acutely toxic micronized powder in both species. Despite its lower overall percentage vanadium content, a liquid aerosol of VOSO4 was more toxic than the V2O5 particles in mice, but not in rats. These data suggest that an interaction of characteristics, i.e., bioavailability, solubility and oxidation state, as well as species sensitivity, likely affect the toxicity potential of vanadium compounds. Based on clinical observations and gross necropsy findings, the lung appeared to be the target organ for all compounds. The level of hazard posed will depend on the specific chemical form of the vanadium. Future work to define the inhalation toxicity potential of vanadium compounds of various oxidation states after repeated exposures will be important in understanding how the physico-chemical and biological characteristics of specific vanadium compounds interact to affect toxicity potential and the potential risks posed to human health.

Do current FIFRA testing guidelines protect infants and children? Lead as a case study. Federal Insecticide, Fungicide, and Rodenticide Act.

With implementation of the Food Quality Protection Act (FQPA), questions have been raised concerning the adequacy of current guideline testing under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) to assure compliance with FQPA. Specifically, there have been questions about whether FIFRA testing is adequate to assure protection of the most sensitive individuals in the population that might be exposed to FIFRA-regulated chemicals. In most cases, concern has been expressed about protection of infants and children. One way to examine the adequacy of current testing is to retrospectively examine data generated from tests similar to, or the same as, standard FIFRA tests on chemicals with known effects on developing organisms, effects that occur at doses below the effect level for adults. Lead is an example of a compound that has been widely studied and where infants and children have been shown to be more susceptible than adults to certain types of lead-induced toxicity. A review of the available animal toxicity studies on lead compounds, studies that were similar in design to testing required under FIFRA, was performed and the adequacy of the studies for setting human exposure levels that would be protective of child and infant health was assessed. The assessment demonstrated that current guideline testing under FIFRA would have resulted in setting of an exposure level in humans that is below the current regulatory action level for lead, without the use of an additional FQPA safety factor.

MK-677, an orally active growth hormone secretagogue, reverses diet-induced catabolism.

The reversal of diet-induced negative nitrogen balance by GH suggests a possible therapeutic role for GH treatment in catabolic patients. A double-blind, randomized, placebo-controlled, two-period cross-over study was designed to investigate whether MK-677, an orally active nonpeptide mimic of GH-releasing peptide, can reverse diet-induced protein catabolism. Eight healthy volunteers (ages 24-39 yr) were calorically restricted (18 kcal/kg.day) for two 14-day periods. During the last 7 days of each diet period, subjects received either oral MK-677 25 mg or placebo once daily. There was a 14- to 21-day washout interval between periods. During the first week of caloric restriction (i.e. diet alone), daily nitrogen losses were similar for both treatment groups (mean +/- SE; MK-677 group -2.67 +/- 0.40 g/day vs. placebo group -2.83 +/- 0.26 g/day). During the second week (diet and study drug), mean daily nitrogen balance was 0.31 +/- 0.21 g/day in the MK-677 treatment group compared with -1.48 +/- 0.21 g/day in the placebo group (P < 0.01). MK-677 improved nitrogen balance integrated over the 7 days of treatment; area under the curve day 8-14 nitrogen balance response was +2.69 +/- 5.0 (SE) for MK-677 and -8.97 +/- 5.26 g.day for placebo (P < 0.001). MK-677 produced a peak GH response of 55.9 +/- 31.7 micrograms/L after single dose (day 1 of treatment) and 22.6 +/- 9.3 micrograms/L after a week of dosing compared with placebo treatment peak GH values of approximately 9 (treatment day 1) and approximately 7 micrograms/L (treatment day 7). Following the initial 7-day caloric restriction, insulin-like growth factor-I (IGF-I) declined from 232 +/- 25 to 186 +/- 19 ng/mL in the MK-677 group and from 236 +/- 19 to 174 +/- 23 ng/mL in the placebo group. Mean IGF-I concentration increased significantly during MK-677 to 264 +/- 31 ng/mL (mean for the last 5 days of treatment) compared with 188 +/- 19 ng/mL with placebo (P < 0.01). No significant difference in IGF binding protein-2 was found between the MK-677 and placebo treatments. However, the mean in IGF binding protein-3 for the last 5 days of MK-677 treatment was also significantly increased to 3273 +/- 330 ng/mL (mean +/- SE) compared with placebo 2604 +/- 253 ng/mL (P < 0.01). Neither the serum cortisol nor the PRL response was significantly greater after 7 days of MK-677 dosing compared with 7 days of placebo. MK-677 (25 mg) was generally well tolerated and without clinically significant adverse experiences. In conclusion, MK-677 reverses diet-induced nitrogen wasting, suggesting that if these short-term anabolic effects are maintained in patients who are catabolic because of certain acute or chronic disease states, it may be useful in treating catabolic conditions.

The pharmacokinetics of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) in Sprague-Dawley rats.

Using adult male Sprague-Dawley rats, we examined the blood protein binding and pharmacokinetics of the potent phencyclidine (PCP) receptor ligand 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP). The average percentage of unbound [3H]TCP in rat serum was 42 +/- 6% and the [3H]TCP blood to plasma ratio was 0.98 +/- 0.03 (mean +/- SD, n = 5 in both studies). For the pharmacokinetic studies, [3H]TCP and 1 mg/kg unlabeled TCP were administered as an iv bolus dose. The average [3H]TCP elimination half-life was 2.1 hr. In contrast, total radioactivity in the plasma had a much longer half-life, suggesting much slower metabolite elimination. The average distribution volumes were 27 +/- 17, 15.6 +/- 6.2, and 5.6 +/- 3.0 liters/kg for V beta, Vss, and Vc, respectively. Total body and renal clearance values were 132 +/- 45 and 1.1 +/- 0.4 ml/min/kg, respectively. When TCP pharmacokinetic parameters were compared to PCP pharmacokinetic data in rats from a previous study, a strikingly similar pharmacokinetic profile was found. These data indicated that TCP and PCP are equivalent, from a pharmacokinetic point of view, and that the higher pharmacological potency of TCP over PCP is probably due to receptor-mediated differences.

Cardiovascular and lethal effects of cocaine in anesthetized dogs and guinea-pigs.

Cardiovascular and lethal effects of i.v. cocaine infusion were studied in anesthetized mongrel dogs and Hartley guinea-pigs. Dogs were anesthetized with enflurane (2.25 vol%) or urethane (1500-1700 mg/kg i.v.) and guinea-pigs with urethane (1000 mg/kg i.p.). The rate of i.v. cocaine infusion was 0.91 mg/kg/min for dogs and 1.9 mg/kg/min for guinea-pigs. In spontaneously breathing dogs and guinea-pigs, death occurred by respiratory arrest, while in artificially ventilated dogs, the terminal stage of cocaine intoxication was peripheral vascular failure. Disturbances in cardiac rhythm (AV-dissociation, ectopic pacemaking activity or irregular rate) were not observed unless mean arterial blood pressure fell to levels inadequate for coronary perfusion. All animals showed an increase in the atrio-ventricular conduction interval (PR-interval). Cardiac arrest (fibrillation or standstill) was never the primary cause of death. Sympathomimetic actions of cocaine, as monitored by heart rate (HR) and mean arterial blood pressure (MAP), were not observed in dogs, while guinea-pigs exhibited only a consistent rise in MAP which was not accompanied by other signs of sympathetic activity. Changes in body temperature were not observed in either species. It is concluded that in anesthetized animals, the predominant cardiovascular and lethal effects of cocaine are the result of its local anesthetic (membrane-stabilizing) action, and that the contribution of its sympathomimetic effects, due to inhibition of neuronal uptake, is masked by its local anesthetic properties.

Effects of morphine pretreatment on cocaine cardiotoxicity in anesthetized guinea-pigs.

Cardiovascular and lethal effects of intravenous cocaine infusion were compared in guinea-pigs pretreated chronically with morphine or saline. Alzet minipumps filled with either morphine solution (30 micrograms/kg/hr for 6 days) or isotonic saline were implanted subcutaneously in animals 6 days before cocaine infusion. On the morning of the 7th day, animals were anesthetized with urethane (1000 mg/kg i.p.) and an i.v. infusion of cocaine was begun at a rate of 1.9 mg/kg/min. All animals were spontaneously breathing room air throughout the experiments. Death occurred by respiratory arrest in all animals; however, there was a significant decrease in the time to respiratory arrest in the morphine-pretreated guinea-pigs. All animals showed a gradual increase in the atrio-ventricular conduction interval (PR-interval) and in mean arterial pressure up until the time that respiration ceased. There were no disturbances in cardiac rhythm before respiratory arrest. This suggests that the predominant cardiovascular and lethal effects of cocaine in urethane-anesthetized guinea-pigs are the result of its local anesthetic (membrane stabilizing) action, and that morphine potentiates this effect of cocaine, but does not alter the pattern of cocaine toxicity.

ANF receptors: distribution and regulation in central and peripheral tissues.

Atrial natriuretic factor is a recently-discovered family of biologically active peptides produced in, stored and secreted by mammalian atria. ANF exerts a wide variety of actions in the periphery as well as within the central nervous system. In general, these actions are directed toward the maintenance of body fluid and electrolyte balance and regulation of arterial blood pressure. In a fashion similar to that of many other hormonal systems, the actions of ANF in various target tissues appear to be mediated by at least one class of specific receptors. However, while the biosynthesis and biological actions of ANF have been extensively investigated, little research has been focused on ANF receptor systems. In this article, we will provide an overview of current literature regarding the distribution and binding characteristics of receptor sites for ANF in peripheral and central target tissues. In addition, we will consider factors involved in the regulation and alteration of ANF receptor sites in various tissues. Finally, a brief discussion of the emerging concept of ANF and angiotensin II as mutual antagonists in body fluid homeostasis and cardiovascular regulation will be offered.

Regulation of binding sites for atrial natriuretic factor (ANF) in rat brain.

In two experiments, binding sites for atrial natriuretic factor (ANF) were studied in discrete areas of rat brain by quantitative autoradiography. In the first experiment, the maximum binding capacity of 125I-ANF was reduced significantly in the subfornical organ and choroid plexus of 4 and 14 week old spontaneously hypertensive (SHR) rats compared to aged-matched Wistar-Kyoto (WKY) normotensive controls. In contrast, the maximum binding capacity of 125I-ANF in the area postrema was similar for young and adult SHR and WKY rats. The second experiment involved a comparison of brain ANF binding sites in Long-Evans control rats and Brattleboro rats with inherited diabetes insipidus. The maximum binding capacity of 125I-ANF was significantly greater in the subfornical organ of Brattleboro rats compared to Long-Evans controls. However, no strain differences occurred for 125I-ANF binding in the choroid plexus or area postrema. These findings indicate that the number of ANF binding sites in discrete areas of rat brain may be influenced in a highly selective fashion by alterations in body fluid homeostasis (i.e., hypertension or diabetes insipidus). Changes in brain ANF binding sites within circumventricular areas may involve central as well as peripheral sources of ANF-related peptides.

Increases in CSF norepinephrine associated with the onset of digoxin-induced arrhythmias.

The present study utilized a time course of CSF catecholamine concentrations during peripheral digoxin administration in vivo to provide a clarification of central catecholaminergic mechanisms involved in digitalis cardiotoxicity. A continuous peripheral digoxin infusion produced significant increases in CSF norepinephrine just prior to arrhythmogenesis in anesthetized dogs. Phentolamine pretreatment significantly increased the arrhythmogenic and lethal doses of digoxin. These results suggest that activation of central noradrenergic neurons may be the initiating factor in digoxin-induced arrhythmogenesis.

Localization of angiotensin II receptors along the anteroventral third ventricle area of the rat brain.

Autoradiographic techniques were utilized to localize and to quantify angiotensin II (ANG) binding sites in rat forebrain. Specific, localized ANG binding sites were demonstrated in midline sagittal sections, corresponding to the entire anteroventral third ventricle (AV3V) area, including the nucleus preopticus medianus and the subependymal area of the anterior third ventricle from the nucleus preopticus medianus to the organon vasculosum laminae terminalis. A continuous band of ANG receptors extended dorsally from the nucleus preopticus medianus along the subependymal area of the third ventricle to the organon subfornicalis. Scatchard analysis performed with consecutive sections from single animals revealed a single class of high-affinity ANG receptors in both the organon subfornicalis and the organon vasculosum laminae terminalis. In addition, ANG receptors were localized in areas anatomically and physiologically related to the AV3V area, including the nuclei paraventricularis and periventricularis and the eminentia mediana. These results support the idea that ANG may act as both a hormone and a neurotransmitter in the central regulation of fluid balance and cardiovascular function, and suggest that the circumventricular organs are the most likely sites for an interaction between the peripheral and central ANG systems.

Central dopamine receptors and their role in digoxin-induced cardiotoxicity in the dog.

The role of the dopaminergic system in digoxin-induced cardiotoxicity has been examined. Specific dopaminergic agonists and antagonists were administered into the ventriculocisternal system of pentobarbitone-anaesthetized dogs before systemic administration of digoxin. Pretreatment with apomorphine, a specific dopamine agonist, did not significantly alter the arrhythmogenic or lethal doses of digoxin. However, the digoxin-induce increase in CSF noradrenaline was decreased significantly in apomorphine-pretreated animals. Pretreatment with pimozide, a specific dopamine antagonist, significantly decreased the arrhythmogenic dose of digoxin but did not alter the lethal dose. As with apomorphine, pimozide-pretreated animals accumulated significantly less noradrenaline in CSF compared with control dogs. These results suggest that dopamine receptors are not directly related to the cardiotoxic actions of digoxin. However, dopaminergic receptors may influence the balance of central catecholaminergic systems that influence the peripheral cardiovascular system.

Quantitative autoradiographic analysis of somatostatin binding sites in discrete areas of rat forebrain.

Somatostatin has been localized in several hypothalamic and extrahypothalamic brain regions where it may function as a classical neurotransmitter or as a modulator of neural activity. In the present study, somatostatin binding sites were studied by incubation of coronal sections of rat forebrain with 125I-Tyr1-somatostatin, Ultrofilm autoradiography, computerized microdensitometry and comparison with 125I standards. Highest concentrations of somatostatin binding sites (fmol/mg protein) were found in the claustrum (151), basolateral nucleus of the amygdala (90), deep layers of the cerebral cortex (61), lateral olfactory nuclei (58), CA1 and CA2 areas of hippocampus (57), medial and lateral septal nuclei (54), and the medial habenula (44). Scatchard analysis of individual forebrain areas with high densities of somatostatin binding sites was also performed. Regulation of brain somatostatin binding sites may be studied as one approach to examining the involvement of central somatostatin pathways in various physiological and behavioral states.

Binding sites for atrial natriuretic factor (ANF) in brain: alterations in Brattleboro rats.

Binding sites for atrial natriuretic factor (ANF-28) were analyzed in discrete brain areas of Brattleboro rats with hereditary diabetes insipidus and Long-Evans (LE) controls by quantitative autoradiography. The maximum binding capacity (Bmax) and affinity constant (Ka) for 125I-ANF-28 were elevated significantly in the subfornical organ of Brattleboro rats compared to matched LE controls. In contrast, values for Bmax and Ka for 125I-ANF-28 binding in choroid plexus and area postrema were similar for rats of the two strains. These findings are consistent with a selective upregulation of ANF-28 binding sites in the subfornical organ of Brattleboro rats which exhibit a profound disturbance in body fluid homeostasis. These alterations in ANF-28 binding sites in the subfornical organ may represent a compensatory response to the absence of vasopressin in the Brattleboro rat.

Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography.

Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM 125I-[Sar1]-angiotensin II, [3H]-Ultrofilm autoradiography, computerized microdensitometry and comparison with 125I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with 125I-[Sar1]-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.

Angiotensin II binding sites in the anteroventral-third ventricle (AV3V) area and related structures of the rat brain.

Angiotensin II binding sites were localized in the rat brain by incubation of sagittal sections with 125I-[Sar1]-angiotensin II, followed by [3H]Ultrofilm autoradiography. Binding sites were localized in the subfornical organ and the entire anteroventral-third ventricle (AV3V) area, including the subependymal area of the anterior third ventricle, the median preoptic nucleus and the organon vasculosum laminae terminalis. Binding sites were also present in the paraventricular and periventricular nuclei and in the median eminence. These findings support the hypothesis of an extensive involvement of angiotensin in the neuronal circuits connecting the subfornical organ, the AV3V area, the paraventricular nucleus and the median eminence, and in the regulation of fluid balance, blood pressure and pituitary secretion.

Quantitative distribution of angiotensin-converting enzyme (kininase II) in discrete areas of the rat brain by autoradiography with computerized microdensitometry.

We report the localization of angiotensin-converting enzyme (kininase II, EC 3.4.15.1) in discrete nuclei and areas of the rat brain by a quantitative autoradiographic technique using image processing coupled to computerized microdensitometry, after incubation of brain sections with the specific converting enzyme inhibitor [125I]351A. High angiotensin-converting enzyme levels are present in circumventricular organs (organon subfornicalis and area postrema), the choroid plexus, and extrapyramidal areas (nucleus caudatus, globus pallidus and substantia nigra) with intermediate levels in selected hypothalamic, septal, habenular and brainstem nuclei. Our results support the idea that angiotensin II could be formed in specific brain areas, both outside and inside the blood-brain barrier. In other brain structures, such as the extrapyramidal areas, kininase II could be involved in the processing or metabolism of other brain peptides.

Binding of angiotensin and atrial natriuretic peptide in brain of hypertensive rats.

Atrial natriuretic peptides, produced in the mammalian cardiac atrium, are released into the general circulation and may be actively involved in the control of blood pressure and in fluid homeostasis as antagonists of the peripheral angiotensin system. Certain cardiovascular effects of atrial natriuretic peptides may be centrally mediated, as binding sites for atrial natriuretic factor (8-33) (ANF) have been localized to the subfornical organ. This circumventricular structure lacks a blood-brain barrier and is therefore accessible to circulating peptides. It contains large numbers of angiotensin II (AII) binding sites, and has been suggested as the main central site of action for circulating AII in the regulation of blood pressure and fluid metabolism. Here we have studied binding sites for rat atrial natriuretic peptide(6-33) (rANP) and AII in the brains of spontaneously (genetic) hypertensive rats (SHR) and their normotensive controls, Wistar Kyoto (WKY) rats, by quantitative autoradiography. Binding sites for both peptides were highly localized in the subfornical organ. The number of rANP binding sites was decreased in the subfornical organ of both young (4 weeks old) and adult (14 weeks old) SHR compared with age-matched normotensive controls. Conversely, the number of AII binding sites was higher in both young and adult SHR compared with WKY rats. Our results suggest a central role for rANP and AII in genetic hypertension; they may act as mutual antagonists in brain areas involved in control of blood pressure and fluid regulation.

Forebrain binding sites for atrial natriuretic factor: Alterations in spontaneously hypertensive (SHR) rats.

Binding sites for atrial natriuretic factor (ANF-28) were studied in forebrain areas of spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) normotensive male rats by quantitative autoradiography. The maximum binding capacity of [(125)I]ANF-28 was significantly reduced in the subfornical organ and choroid plexus of 4 and 14 week old SHR rats compared to age-matched WKY controls. In contrast, the affinity constant for [(125)I]ANF-28 binding was elevated in the choroid plexus of 14 week old SHR rats. These findings indicate that marked reductions in the number of ANF-28 binding sites occur in weanling SHRs as well as in adult SHRs with elevated arterial blood pressures. Thus, these persistant reductions in forebrain ANF-28 binding sites in SHR rats may contribute to the development and maintenance of this form of experimental hypertension.

Quantitative autoradiographic determination of angiotensin-converting enzyme binding in rat pituitary and adrenal glands with 125I-351A, a specific inhibitor.

We describe a quantitative autoradiographic technique which allows measurement of angiotensin-I-converting enzyme [ACE] (kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) levels in discrete areas of pituitary and adrenal glands in individual animals. Tissue sections were incubated with 125I-351A, a specific ACE inhibitor, and results were obtained with computerized densitometry and comparison to 125I standards. There were high levels of ACE in both the anterior and posterior lobes of the pituitary, with no detectable binding in the intermediate lobe. The maximum binding capacity (Bmax) was 920 +/- 62 fmol/mg protein for the anterior pituitary and 1162 +/- 67 fmol/mg protein for posterior pituitary. The binding affinity constant (Ka) was 0.95 +/- 0.11 X 10(9) M-1 and 1.20 +/- 0.19 X 10(9) M-1 for the anterior and posterior lobes, respectively. In the adrenal gland, there were two distinct areas of specific binding, the adrenal medulla and the adrenal capsule-zona glomerulosa area. The Bmax for the adrenal medulla was 652 +/- 80 fmol/mg protein and 294 +/- 53 fmol/mg protein for the adrenal capsule-zona glomerulosa. The Ka for 351A was 1.04 +/- 0.19 X 10(9) M-1 and 1.74 +/- 0.40 X 10(9) M-1 for medulla and adrenal capsule-zona glomerulosa respectively. The results support the existence of local ANG systems active in both the pituitary and adrenal glands.

Quantitative autoradiographic determination of angiotensin-converting enzyme (kininase II) binding in individual rat brain nuclei with 125I-351A, a specific enzyme inhibitor.

We report the localization and characterization of angiotensin-converting enzyme (kininase II) in discrete nuclei from individual rat brains by a quantitative autoradiographic technique coupled to computerized microdensitometry. The enzyme was quantitated by incubation of 16-micron-thick brain sections with 0.07-2 nM of the converting enzyme inhibitor 125I-351A and comparison to 125I-standards. This technique can be applied to the study of other enzymes in single rat brain nuclei.